Hepatocellular carcinoma (HCC), a malignant tumor of the liver, is a major health problem (Llovet et al., Lancet, 362:1907-1917 (2003)). It is the third leading cause of cancer-related death in the world, and its incidence is increasing in Europe and the US. HCC is now the leading cause of death among cirrhotic patients and accounts for 80% to 90% of all liver cancers. It occurs more often in men than women and occurs mostly in people 50 to 60 years old. The disease is more common in parts of Africa and Asia than in North or South America and Europe.
The cause of liver cancer is usually cirrhosis, or scarring of the liver. Cirrhosis may be caused by viral hepatitis, primarily hepatitis B and C, alcohol abuse, hemochromatosis, certain autoimmune diseases of the liver, and a whole host of other diseases that result in chronic inflammation of the liver leading to scarring. While in the U.S. the most common cause for cirrhosis is alcohol abuse, globally, HCV infection is the most prevalent etiology of HCC in Europe, US and Japan (Bosch et al., Gastroenterology, 127(5 Suppl 1):S5-S16 (2004)). HCC is notoriously refractory to treatment. Typically, chemotherapy and radiation treatments are not usually effective but may be used to shrink large tumors so that surgery has a greater chance of success. In some instances, where the tumors are small or slow-growing, surgery or liver transplantation may be successful. However, liver transplant is only effective if such small or slow-growing tumors are diagnosed early in the course of the disease.
Nevertheless, as a result of screening programs in the West and Japan, early diagnosis of HCC is now feasible in 30-60% of cases, enabling the application of curative treatments (Llovet et al., Lancet, 362:1907-1917 (2003); Sangiovanni et al., Gastroenterology, 126(4):1005-1014 (2004)). Simultaneously, however, an increasing number of small nodules of ˜2 cm are detected, which are difficult to characterize by imaging techniques or conventional pathologic examination (Ikai et al., Cancer, 101:796-802 (2004); Bolondi et al., Hepatology, 42:27-34 (2005); Kojiro et al., Liver Transpl., 10(2 Suppl 1):S3-S8 (2004)).
Distinguishing pre-neoplastic lesions, particularly high grade dysplastic nodules (HGDN), from early tumors is an unresolved challenge. Expert hepatopathologists often disagree about the final diagnosis of early tumors, some of which are misclassified as dysplastic nodules, a situation that is estimated to be especially common in non-expert hands (Kojiro et al., Liver Transpl., 10(2 Suppl 1):S3-S8 (2004)). Immunostaining with CD34 and alpha fetoprotein (AFP) has significant diagnostic limitations (Park et al., Am. J. Surg. Pathol., 22:656-662 (1998)). Nonetheless, pathology is considered the gold-standard of diagnosis. Non-invasive radiological criteria have been developed by using state-of-the art imaging techniques, such as contrastenhanced ultrasonography, helical computed tomography or magnetic resonance imaging (Bruix et al., J. Hepatol., 35:421-430 (2001); Burrel et al., Hepatology, 38:1034-1042 (2003)). However, reliable diagnosis is confined almost entirely to tumors exceeding 2 cm in diameter (Lencioni et al., J. Hepatol., 40:162-171 (2004)). Finally, serum biomarkers such as AFP, desgamma-carboxyprothrombin (DGCP) and AFP-L3 fraction are currently not reliable for the early diagnosis of HCC (Bruix et al., Hepatology, 42:1208-1236 (2005); Marrero et al., Gastroenterology, 127(5 Suppl 1):S113-S119 (2004)).
There is a clear distinction between tissue biomarkers and serum biomarkers. Tissue markers should be able to distinguish early HCC from other entities (preneoplastic lesions, cirrhotic tissue and other neoplasms). Eventually, some of them may be further tested as serum markers for surveillance purposes, as defined by the Early Detection Research Network of the National Cancer Institute (Marrero et al., Gastroenterology, 127(5 Suppl 1):S113-S119 (2004)). A variety of genomic studies using genome-wide DNA microarray or quantitative real time reverse-transcriptase polymerase chain reaction (RT-PCR) have attempted to identify markers of early HCC, including heat shock protein 70 (HSP70) (Chuma et al., Hepatology, 37:198-207 (2003)), Glypican-3 (GPC3) (Capurro et al., Gastroenterology, 125:89-97 (2003); Nakatsura et al., Biochem. Biophys. Res. Commun., 306:16-25 (2003); Hippo et al., Cancer Res., 64:2418-2423 (2004)), telomerase reverse transcriptase (TERT) (Smith et al., Cancer Res., 63:859-864 (2003)), serine/threonine kinase 15 (STK6) and phospholipase A2 (PLAG12B) (Paradis et al., Am. J. Pathol., 163:733-741 (2003)). A molecular index including a 13-gene set has also been proposed (including TERT, TOP2A and PDGFRA) (Nam et al., Hepatology, 42:809-818 (2005)). More recently a microarray-generated signature of 120 genes was reported to discriminate between dysplastic nodules and HCC in HBV patients (Paradis et al., Hepatology, 41:40-47 (2005)). Proteomic studies in tissue have not identified informative HCC markers so far (Borzio et al., J. Hepatol., 39:208-214 (2003)).
A major limitation of these studies, however, has been the comparison between the gene expression of cancer with non-tumoral cirrhotic tissue. Data regarding gene expression in dysplastic nodules and early HCC is scarce, and direct comparisons are lacking in HCV patients. Overall, an ideal candidate set of genes has not been identified, and none of the reported genes or signatures is accepted as a molecular marker in standardized guidelines of HCC management (Bruix et al., J. Hepatol., 35:421-430 (2001); Bruix et al., Hepatology, 42:1208-1236 (2005)). Distinction between pre-neoplastic nodules and early small tumors has critical clinical implications. According to the management guidelines of HCC in Europe and the US (Bruix et al., J. Hepatol., 35:421-430 (2001); Bruix et al., Hepatology, 42:1208-1236 (2005)), dysplastic lesions should be followed by regular imaging studies, since approximately one-third of them will develop a malignant phenotype, with the remaining nodules either disappearing or remaining stable for years (Terasaki et al., Gastroenterology, 115:1216-1222 (1998); Kojiro et al., Semin. Liv. Dis., 25:133-142 (2005)). Malignant transformation from low-grade dysplastic nodules is less evident, although these are the precursors of HGDN (Llovet et al., Semin. Liver Dis., 25:181-200 (2005)). On the other hand, early small tumors are the ideal targets for curative treatments such as resection, transplantation and percutaneous ablation that can provide median survivals exceeding 60 months (Llovet et al., Lancet, 362:1907-1917 (2003); International Working Party, Hepatology, 17:27-35 (1995)). Thus, there is an urgent need to identify better tools to characterize these lesions. In the absence of addressing this need, the cost-effectiveness of the recall policies applied within surveillance programs will be significantly undermined.